小鼠白介素10(IL-10)ELISAKit

1、AMEKOTEL:+86-656775338FAXE-mail: lianshuo试剂盒使用说明书AE90224MUAMEK6Onlv use for reM&rrb For life srience小鼠白细胞介素10(IL-10)幅联免疫检测使用前仔细阅读本说明书。本酶联免疫试剂盒是基于双抗体夹心技术原理, 来检测小鼠白细胞介素10(IL-10),只能用于研究用途，不得用于医学诊断。用 途：用于小鼠血清、血浆及相关液体样本中白细胞介素10(IL-10)测定。工作原理本试剂盒采用的是生物素双抗体夹心酶联免疫吸附法(ELIS

2、A)测定样品中小鼠白细胞介素10(IL-10)水平。向预先包被了小鼠白细胞介素10(IL-10)单克隆抗体的酶标孔中加入白细胞介素 10(IL-10),温育；温育后，加入生物素标记 的抗IL-10抗体。再与链霉亲和素-HRP结合，形成免疫复合物，再经过温育和 洗涤，去除未结合的酶，然后加入底物A、B,产生蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅与样品中小鼠白细胞介素10(IL-10)的浓度呈正相关。试剂盒组成试剂盒组成48孔配置96孔配置保存说明书1份1份封板膜2 片（48）2 片（96）密封袋1个1个酶标包被板1X481X962-8 C保存标准品400 ng/L0.5ml X 1 瓶

3、0.5ml X 1 瓶2-8 C保存标准品稀释液3ml x 1 瓶6ml x 1 瓶2-8 C保存链霉亲和素-HRP3 ml x 1 瓶6 ml x 1 瓶2-8C保存生物素标记的抗IL-10抗体0.5ml X 1 瓶1 ml x 1 瓶2-8 C保存显色剂A液3 ml x 1 瓶6 ml x 1 瓶2-8 C保存显色剂B液3 ml x 1 瓶6 ml x 1 瓶2-8 C保存终止液3ml x 1 瓶6ml x 1 瓶2-8 C保存浓缩洗涤液（20ml X 20 倍）X 1 瓶（20mlX30倍）X 1 瓶2-8 C保存需要而未提供的试剂和器材1 . 37 c恒温箱。2 .标准规格酶标仪。3

4、.精密移液器及一次性吸头4 .蒸储水，5 . 一次性试管6 .吸水纸注意事项1.从2-8 C取出的试剂盒，在开启试剂盒之前要室温平衡至少 30分钟。酶标包 被板开封后如未用完，板条应装入密封袋中保存。2,各步加样均应使用加样器，并经常校对其准确性，以避免试验误差3 .严格按照说明书的操作进行，试验结果判定必须以酶标仪读数为准.4 .为避免交叉污染，要避免重复使用手中的吸头和封板膜。5 .不用的其它试剂应包装好或盖好。不同批号的试剂不要混用。保质前使用。6 .底物B对光敏感，避免长时间暴露于光下。洗板方法手工洗板方法：甩掉酶标板内的液体；在实验台上铺垫几层吸水纸，酶标板朝下 用力拍几次；将稀释后

5、的洗涤液至少 0.35ml注入孔内，浸泡1-2分钟。根据需 要，重复此过程数次。自动洗板：如果有自动洗板机，应在熟练使用后再用到正式实验过程中标本要求1 .不能检测含NaN3勺样品，因NaN3卬制辣根过氧化物酶的（HRP活性。2 .标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。若不能马上进行试验，可将标本放于-20 C保存，但应避免反复冻融。操作程序1 .标准品的稀释：（本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀释。）200 ng/L5号标准品120 l的原倍标准品加入120仙l标准品稀释液100 ng/L4号标准品120仙l的5号标准品加入120仙l标

6、准品稀释液50 ng/L3号标准品1201的4号标准品加入120（i l标准品稀释液25ng/L2号标准品1201的3号标准品加入120（i l标准品稀释液12.5 ng/L1号标准品1201的2号标准品加入120（i l标准品稀释液2 .根据待测样品数量加上标准品的数量决定所需的板条数。每个标准品和空白孔建议做复孔。每个样品根据自己的数量来定，能使用复孔的尽量做复孔。3 .加样：1）空白孔，空白对照孔不加样品，生物素标记的抗 IL-4抗体，链霉 亲和素-HRP,只加显色剂A&B和终止液，其余各步操作相同；2）标准品孔： 加入标准品50ul ,链霉亲和素-HRP50U1（标准品中已事先

7、整合好生物素抗体， 故不加）:3）待测样品孔：加入 样本40ul、然后各加入 抗IL-4 抗体10ul、 链霉亲和素-HRP50U1,盖上封板膜，车5轻震荡混匀，37c温育60分钟。4 .配液：将30倍浓缩洗涤液用蒸储水30倍稀释后备用。5 .洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置 30秒后 弃去，如此重复5次，拍干。6 .显色：每孔先加入显色剂 A50ul,再加入显色剂B50p 1,轻轻震荡混匀，37c 避光显色15分钟.7 .终止：每孔加终止液50口，终止反应（此时蓝色立转黄色）。8 .测定：以空白空调零，450nm波长依序测量各孔的吸光度(ODfi)。测定应 在加终止

8、液后10分钟以内进行。9 .根据标准品的浓度及对应的 OD值计算出标准曲线的直线回归方程，再根据 样品的OD值在回归方程上计算出对应的样品浓度。也可以使用各种应用软 件来计算。操作程序总结:检测范围：6ng/L-200 ng/L。保存：2-8 C。有效期：6个月(2-8 C)。Mouse IL-10 ELISA KitInstructionThis kit is only for scientific research, and shall not be used as a clinical diagnosis of use.PurposeThis kit allows for the det

9、erminationof IL-10 concentrationsn Mouseserum, cell culture supernatant, and other biological fluids.PrincipleThe kit assay Mouse-10 level in the samp leadd MousdL-10 antibody to microtiter platewells, after Incubating,add Biotinylated ant1 0-antibody , then Combined Streptavidin-HRP, become complex

10、, after Incubating and washing Completely, Add TMB substrate solution,TMB substrate becomes blue color, reaction is terminated by the addition of a sulphuric acid solution and the color changeis measuredspectrophotometricallyt a wavelengthof 450 nm. The concentration of IL-10 in the samples is then

11、determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kitMaterials provided with th kite48determinations96 determinationsStorageUser manual11Closure plate membrane!22Sealed bags11Microelisa stripplate112-8 CStandard 400 ng/L0.5ml 1Xbottle0.5ml 1Xbottle2-8

12、CStandard diluent3ml 1 bottle6ml 1 bottle2-8 CStreptavidin-HRP3ml 1 bottle6ml 1 bottle2-8 CBiotinylated anti TL-10-antibody0.5ml 1Xbottle1ml 1 bottle2-8 CChromogen Solution A3ml 1 bottle6ml 1 bottle2-8 CChromogen Solution B3ml 1 bottle6ml 1 bottle2-8 CStop Solution3ml 1 bottle6ml 1 bottle2-8 Cwash s

13、olution(20ml x 20 folcD x 1bottle(20mlx 30 folcD x 1 bottle2-8 CMaterials required but not supplied1.37 C incubator2. Standard microplate reader(450nm)3. Precision pipettes and Disposable pipette tips.4. deionized water.5. Disposable Test tube6. Absorbent paperImportant notes1. The kit takes out fro

14、m the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.2. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error.3. Please

15、according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.4. Use new disposal plastic pipette tips and Closure plate membrane for each standard, in order to avoid cross contamination.5. Do not mix reagents with those from other lots.6. T

16、he substrate evade the light preservationSpecimen requirements1 . extract as soon as possibleafter Specimencollection,andaccordingto the relevant literature,and shouldbe experimentas soon as possibleafter the extraction .If it cant, specimen can be kept in - 20 to preserve, Avoid repeated freeze-tha

17、w cycles.2 . Can' t detect the sample which conaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Diloteinal density Standard as follow table:200 ng/L5 Standard120 dOriginal density Standard120Standard diluent100 ng/L4 Standard120 d5 Standard120 d Standard diluent50 ng

18、/L3 Standard120 d4 Standard120 日Standard diluent25 ng/L2 Standard120 d3 Standarc+120 口Standard diluent12.5 ng/L1 Standard120 d2 Standard+120Standard diluent3 .according testing Sample numberSetOne how many wells nedd, Standard and blank suggested Do holes.4 .add sample 1) blank wells(blank compariso

19、n wells don ' t addtsnymped anti-IL-4 -antibody and Streptavidin-HRP ,other each step operation is same); 2) Standard wells:add Standard05仙 and Streptavidin-HRP) 5 3) testing Sample wells: add sample,thenadd antHL-4 -antibody 1(i,lStreptavidin-HRF05L Closing plate with Closure platemembrane ,inc

20、ubate for 60 min aC374.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5 .washing Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6 .color Add Chromogen Solution